PROJECT SUMMARY/ABSTRACT Diabetes affects over 30 million people in the United States and costs a staggering $327 billion a year in direct medical costs and lost productivity. Diabetes adversely affects blood vessels as hyperglycemia and insulin resistance are key players in the development of atherosclerosis, peripheral neuropathy, retinopathy, and peripheral artery disease. Peripheral artery disease is a chronic condition where fatty deposits called plaques build up in the arteries to the legs, resulting in ulcerations and infections, which precedes 85% of diabetes- related amputations. Our goal is to understand how chronic hyperglycemia impairs vascular endothelial cell function to identify molecular targets that will form the basis for new therapeutic approaches to treat ulcerations and the other vascular complications of diabetes. Vascular endothelial growth factor receptors 2 and 3 (VEGFR2/3) are critical regulators of blood vessel growth or angiogenesis. These receptors are significantly reduced in the vascular endothelium of diabetic patients, resulting in inadequate angiogenesis. In our previously funded application, we showed that diabetic conditions induced expression of autophagosome proteins and promoted degradation of VEGFR2/3. In particular, we identified the protein Unc-51-like autophagy activating kinase 1 (Ulk1) as an important inhibitor of angiogenesis by stimulating autophagosome formation causing selective degradation of VEGFR2/3. Loss of endothelial Ulk1 elevated VEGFR2/3 levels and enhanced angiogenic responses such as endothelial cell proliferation, migration, and tube formation. In this competing renewal application, we present compelling preliminary data demonstrating the Forkhead box O1 transcription factor (FoxO1) controls expression of endothelial Ulk1 in both in vivo and in vitro diabetic model systems and that deficiency of endothelial FoxO1 inhibits autophagosome formation. We also show the noncoding RNA miR183-3p inhibits endothelial FoxO1 expression and the deficiency of epsin 1 and 2 adaptor proteins promotes FoxO1 ubiquitination and degradation in diabetes. These findings strongly suggest that targeting FoxO1 to protect VEGFR2/3 from degradation may represent a novel therapeutic strategy to prevent inadequate vascularization in diabetic ulcers. In view of that, we will investigate the following Specific Aims using unique mutant mice as well as in vitro models of diabetes: 1) determine the molecular mechanisms underlying FoxO1-mediated inhibition of neovascularization in diabetes, 2) determine the molecular mechanisms regulating FoxO1 activity in the diabetic endothelium, and 3) determine the therapeutic potential of targeting FoxO1 by genetic deletion or miR183-3p-mediated inhibition in diabetic angiogenesis. Our findings will enhance understanding of the cellular mechanisms behind VEGFR2/3 loss and activation of FoxO1 in regulating blood vessel damage in diabetes. We anticipate that therapies targeting FoxO1 may be useful for restoring peripheral angiogenesis to ameliorate the vascular complications associated with diabetes.